The activation domains of p53 and NF-κB p65 contain a FXXΦΦ motif. (A) Amino acid sequences of the activation domains of VP16, p53, and p65. FXXΦΦ sequences are indicated by boxes. (B) In vitro protein–protein interaction assays. Binding of TAF_1–140 to GST-VP16_452–490 (lane 1), GST-VP16_469–485 (lane 2), GST-p53_1–52 (lane 3), GST-p53_9–25 (lane 4), GST-p65_522–551 (lane 5), and GST-p65_532–548 (lane 6) is evident. TAF_1–140 is not retained on GST resin (lane 7). The position of TAF_1–140 is indicated by an arrowhead. (C) In vitro transcription assays. Transcriptional activation by activation domains fused with the GAL4 DNA-binding domain was assayed in HeLa nuclear extracts. The reactions contained 2 pmol of the purified proteins and 100 ng of pG5BCAT template containing the adenovirus E1b promoter linked to five GAL4 recognition sites. The products of the transcription reactions were analyzed by primer extension. Lane 1 shows basal transcription without GAL4 proteins. The transcriptional activation by GAL4-VP16_452–490 (lane 3), GAL4-VP16_469–485 (lane 4), GAL4-p53_1–52 (lane 5), GAL4-p53_9–25 (lane 6), GAL4-p65_522–551 (lane 7), and GAL4-p65_532–548 (lane 8) is greater than that observed for GAL4 alone (lane 2).

Transferred NOE (TRNOE) experiments. (A) The amide region of a 200-ms NOESY spectrum of p53_9–25 (2.8 mM) in the presence of TAF_1–140 (0.3 mM). The identities of residues that exhibit NOE cross-peaks in this region are indicated. (B) The αβ region of a 350-ms NOESY spectrum of p53_10–25 (3.5 mM) in the presence of TAF_1–140 (0.3 mM). The identities of key NOEs are indicated. (C) Summary of the NOEs observed from the TRNOE experiments of p53_9–25 and p53_10–25 and comparison between p53_9–25 and VP16_469–485. The thickness of the lines indicates the relative intensities of the NOE cross-peaks. Black coils above the sequences represent the regions over which α-helix induction is observed upon binding to TAF_1–140. The data of VP16_469–485 are from ref. 18.

(A) Helical wheel presentation of residues 18–24 of p53. The residues that mutational studies have shown to be critical for transcriptional activation are indicated by boxes. (B) Substitution of Phe¹⁹ and Trp²³ with Ala in p53_9–25 significantly reduces binding to TAF_1–140 in vitro. It is evident that binding of GST-p53_9–25 (F¹⁹W²³ → AA mutant) to TAF_1–140 (lane 2) is much weaker than that of GST-p53_9–25 [wild type (WT); lane 1]. (C) Transient transfection assays. Expression plasmids encoding GAL4-p53_1–52 and its mutant were transiently transfected into Jurkat cells together with a reporter plasmid in which the production of secreted alkaline phosphatase (SEAP) is under control of an IL-2 promoter carrying five GAL4-binding sites.

Helix disruption greatly impairs TAF binding and transcriptional activation. (A) In vitro protein–protein interaction assays. Asp²¹ at the third position of the FXXΦΦ motif in the p53 activation domain was substituted with helix-breaking Pro. This substitution greatly reduced the binding affinity for TAF_1–140 (compare lanes 1 and 2), whereas substitution of Asp²¹ with Ala had no significant effect on TAF binding (lane 3). TAF_1–140 is not retained on GST resin (lane 4). The position of TAF_1–140 is indicated by an arrowhead. (B) Transient transfection assays. Expression plasmids encoding GAL4-p53_1–52 and its mutants (D²¹ → P and D²¹ → A) were transiently transfected into Jurkat cells together with a reporter plasmid in which the production of secreted alkaline phosphatase (SEAP) is under control of an IL-2 promoter carrying five GAL4-binding sites. Gel mobility-shift analyses of the cell lysates also were performed to assess the expression levels of the mutant proteins. Lane 2 shows a control with lysate of the cells transfected only with the reporter plasmid. In lane 6, an excess of unlabeled probe was used as competitor. The position of the protein–DNA complex is indicated by an arrowhead.

MDM2 discriminates among FXXΦΦ motifs. (A) In vitro protein–protein interaction assays. Binding of GST-p53_1–52 (lane 3) to MDM2_3–109 is evident, and MDM2_3–109 is not retained on GST-VP16_452–490 (lane 1), GST-VP16_469–485 (lane 2), GST-p65_522–551 (lane 5), GST-p65_532–548 (lane 6), and GST (lane 7) resins. MDM2_3–109 binds to GST-p53_9–25 (lane 4) much more weakly than to GST-p53_1–52. (B) Effects of amino acid substitutions and deletion on the interaction of the p53 activation domain with MDM2_3–109 and TAF_1–140 in vitro. The positions of MDM2_3–109 and TAF_1–140 are indicated by arrowheads. (C) Effects of substitution of Trp²³ on transcriptional activation in transfected cells and response to MDM2 coexpression. Increasing amounts of a plasmid encoding human MDM2 were transfected into Jurkat cells cotransfected with an expression plasmid of GAL4-p53_1–52 or its mutants and a reporter plasmid in which the production of secreted alkaline phosphatase (SEAP) is under control of an IL-2 promoter carrying five GAL4-binding sites.